Somatic histone mutations are a hallmark of pediatric high-grade gliomas (pHGGs). Recurrent mutations in the genes encoding for histone variants H3.3 (H3F3A) and H3.1 (HIST1H3B, HIST1H3C) lead to amino acid substitutions at two key residues in the histone tail: lysine to methionine at position 27 (K27M) and glycine to arginine or valine at position 34 (G34R/V). H3.3K27M mutations are associated with distinct clinicopathological characteristics, such as anatomical distribution of tumors carrying these mutations, histological features, age at presentation and survival time. Although great progress has been made in understanding the inhibition of methyltransferases by K27M mutations, additional molecular mechanisms that contribute to the overall poor survival of H3.3K27M pHGG patients may have been overlooked. By DNA sequence analysis, we have recently predicted that the H3.3K27M mutation simultaneously disrupts the K27 codon of the H3F3A gene and the core unit of a DNA binding motif, which is recognized by the DNA-binding protein CCCTC-binding factor (CTCF). Preliminary chromatin immunoprecipitation data in patient derived pHGG cell lines and primary pHGG tumors indicate that CTCF indeed binds the H3F3A wild type, but not the H3.3K27M mutant allele. Based on our compelling preliminary results, we now propose the central hypothesis that the exonic CTCF binding site plays a critical role in the regulation of DNA loops and gene expression, and the disruption of the CTCF binding site by H3.3K27M mutations contributes to gliomagenesis. Toward this objective, we propose: (i) To validate the effects of the mutated CTCF binding site on the disruption of DNA loops and enhancer-mediated misregulation nearby genes in primary pHGG tumors and patient derived pHGGs cell lines (Aim 1); (ii) To validate the relevance of the disrupted exonic CTCF binding site for early transformation leading to pHGGs. We aim to generate targeted induced pluripotent stem cells (iPSCs) by replacing the endogenous H3F3A allele with a wildtype H3.3 sequence (H3.3-CTCF+) fused to an inducible H3.3 version with synonymous nucleotide substitutions (H3.3-CTCF-). Synonymous H3.3-CTCF- substitutions disrupt the CTCF binding site while retaining the wild-type H3F3A amino acid sequence. Targeted isogenic iPSCs will be differentiated into different cell types of the neural lineage, including oligodendroglial precursor cells (OPCs), and assessed for relative changes in proliferation, apoptosis, and differentiation in induced H3.3-CTCF- compared to uninduced H3.3-CTCF+ cells (Aim 2). Upon conclusion, we will have functionally tested the influence of the mutated exonic CTCF binding site on chromosome conformation, gene regulation, and impaired differentiation into OPCs as an additional molecular mechanism of H3.3K27M gliomagenesis.